费斯特共振能量转移
大麻素受体
激酶
磷酸化
大麻素
兴奋剂
化学
生物物理学
细胞内
生物化学
受体
细胞生物学
生物
荧光
量子力学
物理
作者
Ying Liu,Luyao Chen,Hong Zeng,Richard J. Ward,Nan Wu,Li Ma,Xi Mu,Qiulan Li,Yang Yang,Su An,Xuewu Guo,Qian Hao,Tian‐Rui Xu
标识
DOI:10.1016/j.biocel.2018.04.003
摘要
The cannabinoid receptor 1 (CB1) is mainly expressed in the nervous system and regulates learning, memory processes, pain and energy metabolism. However, there is no way to directly measure its activation. In this study, we constructed a CB1 intramolecular fluorescence resonance energy transfer (FRET) sensor, which could measure CB1 activation by monitoring structural changes between the third intracellular loop and the C-terminal tail. CB1 agonists induced a time- and concentration-dependent increase in the FRET signal, corresponding to a reduction in the distance between the third intracellular loop and the C-terminal tail. This, in turn, mobilized intracellular Ca2+, inhibited cAMP accumulation, and increased phosphorylation of the ERK1/2 MAP kinases. The activation kinetics detected using this method were consistent with those from previous reports. Moreover, the increased FRET signal was markedly inhibited by the CB1 antagonist rimonabant, which also reduced phosphorylation of the ERK1/2 MAP kinases. We mutated a single cysteine residue in the sensor (at position 257 or 264) to alanine. Both mutation reduced the agonist-induced increase in FRET signal and structural changes in the CB1 receptor, which attenuated phosphorylation of the ERK1/2 MAP kinases. In summary, our sensor directly assesses the kinetics of CB1 activation in real-time and can be used to monitor CB1 structure and function.
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